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United States Patent |
5,160,669
|
Wallach
,   et al.
|
*
November 3, 1992
|
Method of making oil filled paucilamellar lipid vesicles
Abstract
A new "cold-loading" technique for filling the amorphous central cavity of
paucilamellar lipid vesicles with a water immiscible material has been
developed. Preformed, substantially aqueous filled paucilamellar lipid
vesicles are mixed with the water immiscible material to be encapsulated
under intermediate mixing conditions, thereby replacing the aqueous
solution with the water-immiscible solution. The "cold-loading" technique
is particularly useful for encapsulation of volatiles and heat labile
materials.
Inventors:
|
Wallach; Donald F. H. (Hollis, NH);
Mathur; Rajiv (Nashua, NH)
|
Assignee:
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Micro Vesicular Systems, Inc. (Nashua, NH)
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[*] Notice: |
The portion of the term of this patent subsequent to May 28, 2008
has been disclaimed. |
Appl. No.:
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598120 |
Filed:
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October 16, 1990 |
Current U.S. Class: |
264/4.3; 424/450; 428/402.2 |
Intern'l Class: |
A61K 009/127; B01J 013/20 |
Field of Search: |
264/4.3
428/402.2
424/450
436/829
|
References Cited
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4241046 | Dec., 1980 | Papahadjopoulos et al. | 424/450.
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4247411 | Jan., 1981 | Vanlerberghe et al. | 428/402.
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4297374 | Oct., 1981 | Wess | 514/777.
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4348329 | Sep., 1982 | Chapman | 260/463.
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4356167 | Oct., 1982 | Kelly | 424/450.
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4485054 | Nov., 1984 | Mezei et al. | 264/4.
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4508703 | Apr., 1985 | Redziniak et al. | 424/450.
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4536324 | Aug., 1985 | Fujiwara et al. | 252/311.
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4551288 | Nov., 1985 | Kelly | 264/4.
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4564599 | Jan., 1986 | Janoff et al. | 436/507.
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4608211 | Aug., 1986 | Handjani et al. | 264/4.
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4610868 | Sep., 1986 | Fountain et al. | 424/1.
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4619913 | Oct., 1986 | Luck et al. | 514/2.
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4666711 | May., 1987 | Vanlerberghe et al. | 424/70.
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4695554 | Sep., 1987 | O'Connell et al. | 436/528.
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4772471 | Sep., 1988 | Vanlerberghe et al. | 424/450.
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4814270 | Mar., 1989 | Piran | 435/7.
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4885712 | Dec., 1989 | Bally et al. | 424/450.
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4911928 | Mar., 1990 | Wallach | 424/450.
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4942038 | Jul., 1990 | Wallach | 424/450.
|
5019174 | May., 1991 | Wallach | 424/450.
|
Foreign Patent Documents |
0032578 | Jul., 1984 | EP.
| |
3410602 | Sep., 1984 | DE.
| |
59-106423 | Jun., 1984 | JP.
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61-207324 | Sep., 1986 | JP.
| |
85/01440 | Apr., 1985 | WO.
| |
1539625 | Jan., 1979 | GB.
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2078543A | Jan., 1982 | GB.
| |
2079179A | Jan., 1982 | GB.
| |
2147263A | May., 1985 | GB.
| |
2166107A | Apr., 1986 | GB.
| |
Other References
Bangham et al. (1965) J. Mol. Biol. 13:238-252.
Gregoriadis (1976) The New England Journal of Medicine 295:704-710.
Szoka, Jr. et al. (1978) Proc. Natl. Acad. Sci. USA 75:4194-4198.
Liposomes (Ostro, ed.) 1983, Marcel Dekker, Inc. New York, pp. 246-249.
Philippot et al. (1983) Biochem. Biophys. Acta 734:137-143.
Ribier et al. (1984) Colloids and Surfaces 10:155-161.
Baillie et al. (1985) J. Pharm. Pharmacol. 37:863-868.
"Methodes de preparation des liposomes", Dousset et al. (Puisieux and
Dellattre, Eds.) 1985, Techniques et Documentation La Voisier Paris, pp.
41-72.
"Les niosomes", Handiani-Vila et al. (Puisieux and Dellattre, Eds.) 1985,
Techniques et Documentation La Voisier Paris, pp. 297-313.
Philippot et al. (1985) Biochem. Biophys. Acta 821:79-84.
"Problems technoloqiques poses par l'utilisation des liposomes comme
vecteurs de substances medicamenteuses. Encapsulation, sterilsation,
conservation", Puisieux et al. (Les Liposomes, Eds.) 1985, Techniques et
Documentation La Voisier Paris, pp. 73-113.
Baillie et al. (1986) J. Pharm. Pharmacol. 38:502-505.
|
Primary Examiner: Lovering; Richard D.
Parent Case Text
REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of U.S. patent application Ser.
No. 410,650, filed Sep. 21, 1989 and now U.S. Pat. No. 5,019,174, entitled
"Liposomal Cleaner," which is a continuation-in-part of Ser. No. 157,571,
filed Mar. 3, 1988, now U.S. Pat. No. 4,911,928, issued Mar. 27, 1990,
entitled "Paucilamellar Lipid Vesicles." This application is also a
continuation-in-part of U.S. patent application Ser. No. 443,516, filed
Nov. 29, 1989, also entitled "Paucilamellar Lipid Vesicles," which is a
divisional of the aforementioned Ser. No. 157,571, now U.S. Pat. No.
4,911,928. The disclosures of all the above applications and patents are
incorporated herein by reference.
Claims
What is claimed is:
1. A method of forming a paucilamellar lipid vesicle having a substantially
oil-filled amorphous central cavity comprising the steps of:
preforming a paucilamellar lipid vesicle having an aqueous material in the
amorphous central cavity;
mixing said preformed paucilamellar lipid vesicle with a water immiscible
material to be incorporated into said central cavity under mixing
conditions such that said water immiscible material is incorporated into
said preformed vesicle; and
separating said paucilamellar lipid vesicle from any of said water
immiscible material not incorporated into said central cavity,
whereby said amorphous central cavity of said paucilamellar lipid vesicle
is substantially filled with said water immiscible material.
2. The method of claim 1 wherein an indifferent surfactant is provided in
addition to the lipid used to form said vesicle.
3. The method of claim 2 wherein said indifferent surfactant is
water-soluble.
4. The method of claim 3 wherein said water-soluble indifferent surfactant
is selected from a group consisting of polyoxyethylene sorbitan esters,
sodium dodecyl sulphate, C.sub.12 -C.sub.18 fatty acids, and salts and
mixtures thereof.
5. The method of claim 2 wherein said indifferent surfactant is not water
soluble.
6. The method of claim 1 wherein said water immiscible material to be
incorporated in said amorphous central cavity is volatile or heat labile
at the temperatures used to preform said paucilamellar lipid vesicle.
7. The method of claim 6 wherein said water immiscible material is selected
from a group consisting of diethyltoluamide, flavor oils, fragrance oils,
d-limonene, water immiscible solvents, and mixtures thereof.
8. The method of claim 1 wherein said paucilamellar lipid vesicles have a
non-ionic lipid as the primary bilayer-forming material.
9. The method of claim 1 wherein said paucilamellar lipid vesicles have a
phospholipid as the primary bilayer-forming material.
10. The method of claim 1 wherein said preformed paucilamellar lipid
vesicle contains oil in addition to said aqueous material in said
amorphous central cavity.
Description
BACKGROUND OF THE INVENTION
The present invention relates to a method of forming paucilamellar lipid
vesicles which have amorphous central cavities substantially filled with a
water immiscible material such as an oil. The invention is a
"cold-loading" technique which allows incorporation of volatile and/or
heat labile (heat degraded) materials which could not otherwise be
incorporated into the vesicles.
U.S. Pat. No. 4,911,928 describes a "hot-loading" method of making
paucilamellar lipid vesicles with water immiscible material substantially
filling the amorphous central cavities. The lipid (and any oil or water
immiscible material to be incorporated) is heated to an elevated
temperature, e.g., a liquid or flowable form, so that it can be injected
into an excess of an aqueous phase. This injection of the lipid into the
aqueous phase causes the formation of small lipid micelles (probably
spheroidal) which aggregate upon cooling with turbulent or shear mixing.
The aggregated micelles fuse into vesicles with multiple bilayer shells
surrounding a central, amorphous core. If an oil or a water immiscible
material is also present, both lipid micelles and microemulsion oil
droplets are formed. The microemulsion oil droplets act as nuclei about
which the micelles aggregate, forming an oil-filled amorphous central
cavity of the vesicle surrounded by the lipid bilayers. Preferably, a
small amount of an indifferent surfactant is also included to stabilize
the oil. The term "indifferent surfactant," as used herein, means a
surfactant which will not form lipid vesicles but is able to emulsify the
water immiscible materials to be encapsulated. Indifferent surfactants
include most polyoxyethylene sorbitan ethers (Tweens), sodium dodecyl
sulphate, and C.sub.12 -C.sub.18 fatty acids and their salts such as
sodium oleate. If an indifferent surfactant is not used, a portion of the
wall-forming lipid is cannibalized to stabilize the oil.
Although the "hot-loading" method is effective for a large number of water
immiscible materials, the method is not useful for a variety of important
water immiscible materials which are too volatile or heat labile at the
vesicle forming temperatures. If the "hot-loading" methods are tried for
these thermolabile materials, the majority of the water immiscible
material is volatilized, leaving only a small portion to be incorporated
into the vesicle. These volatile materials include insecticides such as
diethyltoluamide (DEET), certain perfumes and fragrances, flavor oils, as
well as many other materials such as mineral spirits. Since some
fragrances are mixtures, release of one part of the mixture can change the
overall properties dramatically. Further, even certain non-volatiles are
more easily introduced into the amorphous central cavities of vesicles
using the present "cold-loading" technique than the "hot-loading"
technique. For example, the cleaning agent d-limonene can be incorporated
into vesicles at a relatively low concentration using "hot-loading" but a
much higher concentration can be achieved using the "cold-loading"
technique.
Accordingly, an object of the invention is to provide a method of
"cold-loading" the amorphous central cavities of paucilamellar lipid
vesicles with water immiscible materials.
Another objection of the invention is to provide a means of incorporating
volatiles into paucilamellar lipid vesicles.
A further object of the invention is to provide a generalized means of
loading lipid vesicles with oily or water immiscible material which can be
used with phospholipid, ionic, and nonionic lipid materials.
Further objects and features of the invention will be apparent from the
description and the Drawing.
SUMMARY OF THE INVENTION
The present invention features a method of "cold-loading" the amorphous
central cavities of paucilamellar lipid vesicles with water immiscible
materials. The method is particularly important for volatile materials
which cannot be loaded in significant quantities into the central cavities
of paucilamellar lipid vesicles using a "hot-loading" technique.
The method of the invention commences with the formation of paucilamellar
lipid vesicles having substantially aqueous-filled amorphous central
cavities. The vesicles may be made by any classic technique but the
methods and materials disclosed in U.S. Pat. No. 4,911,928 are preferred.
Briefly, these methods require the injection of a flowable lipid, with or
without a small portion of oil, into an excess of an aqueous phase using
shear mixing techniques. The term "shear mixing," as defined in the
aforementioned U.S. Pat. No. 4,911,928, means that the flow of the phases
is equivalent to a relative flow of about 5-50 m/s through a 1 mm orifice.
The resulting vesicles have the amorphous central cavity filled with an
aqueous solution, possibly with some oil included.
After formation of the substantially aqueous-filled paucilamellar lipid
vesicles, they are mixed with the water immiscible material, e.g., an oil,
most preferably a volatile oil, to be incorporated into the amorphous
central cavity under intermediate mixing conditions. The term
"intermediate mixing conditions" means mixing of the preformed vesicles
and the water immiscible material at or near room temperature under gentle
conditions such as vortexing or syringing. Although flow conditions which
yield a shear similar to that used to form the paucilamellar lipid
vesicles initially could be used, it is unnecessary and may, in fact, be
counterproductive.
Following this procedure, the amorphous central cavity of the lipid
vesicles is filled with the water immiscible material, displacing the
aqueous solution. The water immiscible material may act as a carrier for
materials which are soluble or dispersed in it. The paucilamellar lipid
vesicles are then separated from any excess oil, e.g., by centrifugation.
Preferably, an indifferent surfactant is used in the process to stabilize
the water immiscible material. The indifferent surfactant is normally
aqueous soluble and carried in an external aqueous phase but a water
insoluble indifferent surfactant can be incorporated in the amorphous
center or walls of the paucilamellar lipid vesicles before the
intermediate mixing. Preferred indifferent surfactants are selected from
the group consisting of sodium dodecyl sulphate, C.sub.12 -C.sub.18 fatty
acids, Tweens (polyoxyethylene sorbitan esters), and their salts, and
mixtures thereof.
Although the preferred paucilamellar lipid vesicles of the invention have
non-ionic materials such as polyoxyethylene fatty acid esters,
Polyoxyethylene glycerol monostearate, polyoxyethylene steryl alcohols,
and diethanolamides as the wall or bilayer forming lipid, other materials
such as phospholipids, betaines, and other ionic or zwitterion materials
may be used. The invention is particularly preferable for encapsulation of
volatiles or heat labile materials which are not stable liquids at
temperatures where the wall forming lipid is a liquid.
Further aspects and features of the invention will apparent from the
following description.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1a and 1b are illustrations of micelle formation upon injection of a
lipid phase into an excess of aqueous phase such as is used in the
"hot-loading" technique;
FIGS. 2a and 2b are illustrations of micelle and microemulsion formation
upon injection of a lipid and water immiscible material phase into an
excess of aqueous phase such as is used in the "hot-loading" technique;
and
FIG. 3 is a schematic of the endocytosis mechanism suggested for the
"cold-loading" technique of the present invention.
DESCRIPTION OF THE INVENTION
The "cold-loading" method of the present invention is preferable to the
"hot-loading" method where the material to be loaded into the amorphous
central cavity of the vesicles is a volatile or heat labile water
immiscible material. Further, even though the material to be incorporated
in the central cavity may not be volatile, a higher concentration of the
water immiscible material may be loaded using the methods of the present
invention.
FIGS. 1a and 1b illustrate the critical step in the "hot-loading" technique
without oil present, the formation of micelle structures by injection of
the lipid phase into an excess of an aqueous solution. As noted
previously, the micelles aggregate to form the bilayers of the
paucilamellar lipid vesicle.
FIGS. 2a and 2b show the same mechanism with a water immiscible material
added to the lipid. Both micelles and microemulsion oil droplets form.
These microemulsion droplets are the nuclei about which the bilayers of
the lipid vesicle form. Since these microemulsion oil droplets are
necessary in this "hot-loading" technique, clearly a volatile material
which will not form these microemulsions droplets are not appropriate for
the "hot-loading" technique.
FIG. 3 illustrates the most likely mechanism of the "cold-loading"
technique of the present invention. Once the substantially aqueous-filled
paucilamellar lipid vesicles are formed, e.g., using the technique shown
in FIG. 1, they are combined with the cargo material, e.g., the water
immiscible material, preferably in the presence of a low concentration
(approximately 1.5%) of an indifferent surfactant such as sodium dodecyl
sulphate. Droplets of the water immiscible material (stabilized by the
indifferent surfactant) enter the vesicles, presumably by a process
resembling endocytosis.
Although the "cold-loading" technique is most preferred for volatile or
thermolabile materials such as fragrance oils, flavor oils, and certain
lipids or drugs, it is also particularly good for water immiscible
materials which interfere with micelle formation and/or fusion. This
latter group of materials includes diethyltoluamide, d-limonene, and
certain water immiscible solvents such as petroleum distillates and
aromatic solvents such as xylene. These materials, which cannot be
encapsulated in lipid vesicles in any large quantity using the
"hot-loading" techniques, can be incorporated in the amorphous central
cavity of the paucilamellar lipid vesicles using the "cold-loading"
technique of the present invention.
The following Examples will more clearly elucidate the present invention.
EXAMPLE 1
In this Example, aqueous-filled vesicles were made using the methods
described in U.S. Pat. No. 4,911,928 from polyoxyethylene (9) glycerol
monostearate, cholesterol, and a 1.5% solution of Tween 40
(polyoxyethylene 20 sorbitan monopalmitate). Briefly, the patent describes
a technique whereby all of the lipid soluble materials (including any
water immiscible materials if used), are blended together at elevated
temperature until flowability. Normally, this requires a temperature of
60.degree.-80.degree. C. but in some cases as high as 90.degree. C. The
aqueous phase, which includes all the water soluble materials (including
the indifferent surfactant, here the Tween), is also heated. The lipid
phase in then injected into an excess of the aqueous phase through a
moderate shear device and the mixture is sheared until vesicles form.
While a device such as the mixing machine shown in U.S. Pat. No.
4,895,452, the disclosure of which is incorporated herein by reference,
may be used, a pair of syringes connected by a three-way stopcock can
provide shear sufficient for formation of the vesicles. The shear required
is a relative flow of about 5- 50 m/s through a 1 mm orifice. Further
details of this process are described in U.S. Pat. No. 4,911,928. Table 1
lists the formula used to make the vesicles.
TABLE 1
______________________________________
POE (9) glycerol monostearate
20.3 g
Cholesterol 3.5 g
Tween 40 (1.5% solution in water)
75 ml
______________________________________
The preformed vesicles were then mixed with an excess of a water immiscible
material by placing the vesicles in one syringe, an excess of the water
immiscible material which was to act as the cargo in a second syringe, and
the syringes are joined through a three-way stopcock. The solutions were
mixed from one syringe to the other for approximately 40-50 strokes at
ambient temperature. The resulting solution was then centrifuged at 3500
RPM for 30 minutes to separate the unencapsulated water immiscible
material from the lipid vesicles. Table 2 lists the water immiscible
material uptake for a variety of different water immiscible materials. All
values are in ml of water immiscible material/ml vesicle.
TABLE 2
______________________________________
Mineral Oil 1.0 ml/ml
Butyl Cellosolve 0.11 ml/ml
Mineral Spirits 0.18 ml/ml
Isodecyl Benzoate 1.0 ml/ml
Tricresyl Phosphate
1.0 ml/ml
______________________________________
As can be seen, a large number of different materials can be incorporated
at high concentration using this "cold-loading" procedure.
EXAMPLE 2
In this Example, a different wall forming material, polyoxyethylene 2
stearyl alcohol, and a different indifferent surfactant, sodium dodecyl
sulphate (SDS), were used to form the vesicles. The amounts used to
preform the vesicles are shown in Table 3.
TABLE 3
______________________________________
POE (2) Stearyl Alcohol
5.9 g
Cholesterol 2.1 g
1.5% SDS in Water 41.5 ml
______________________________________
The vesicles were formed in the same manner as described in connection with
Example 1. The vesicles were then mixed with an excess of mineral oil
(Drakeol #19) using the same syringe procedure as previously described and
the oil-filled vesicles were separated by centrifugation. The uptake of
mineral oil into the vesicles was greater than 0.7 ml oil/ml vesicle.
EXAMPLE 3
In this Example, a phospholipid, lecithin, was used to form the vesicles.
The lecithin was dissolved in soybean oil, heated until a clear solution
was formed, and then mixed with an excess of water, using the procedure
described in Example 1, to form paucilamellar lipid vesicles. Table 4
shows the amounts of the different components used to form the vesicles.
The vesicles included some oil in the aqueous center.
TABLE 4
______________________________________
Lecithin (98%, Emulpur N-P1
6.4 g
Lucas Meyer, Inc.)
Soybean Oil 6.4 ml
Water 26.0 ml
______________________________________
The preformed phospholipid paucilamellar lipid vesicles were then mixed
with an excess of additional soybean oil using the syringe technique
previously described and centrifuged at 3500 RPM for 30 minutes. The
uptake of the soybean oil in the second processing step was approximately
1 ml oil/ml vesicle. The same procedure has also been used with a 33%
solution of cholesterol oleate in soybean oil being incorporated into the
vesicles. The uptake was at least 0.67 ml/ml vesicle.
EXAMPLE 4
In this Example, additional oil was incorporated into the amorphous center
of nonionic lipid vesicles which already had a small amount of oil
therein. The procedures used were the same as those described in
connection with Example 1 except mineral oil was incorporated into the
heated lipid solution used to form the initial vesicles. Table 5 gives the
ingredients used to preform the vesicles.
TABLE 5
______________________________________
POE (9) Glycerol Monostearate
20.3 g
Cholesterol 3.5 g
Mineral Oil (Drakeol #19)
25.0 ml
1.5% SDS in Water 75.0 ml
______________________________________
After the vesicles were formed, they were mixed using the syringe method
with additional mineral oil and centrifuged at 3500 RPM for 15 minutes to
separate the vesicles from the oil. Uptake of additional mineral oil was
approximately 0.7 ml mineral oil/ml vesicle.
EXAMPLE 5
In this Example, the uptake of DEET (diethyltoluamide) into negatively
charged vessels was tested. DEET interferes with vesicle forming using a
"hot-loading" technique, so insufficient amounts of DEET can be
incorporated into vesicles using the "hot-loading" procedure. Negatively
charged vesicles were formed using the same procedures as described in
Example 1, using the materials shown in Table 6.
TABLE 6
______________________________________
POE (9) Glycerol Monostearate
11.2 g
Cholesterol 1.9 g
Oleic Acid 0.2 g
Tween 40 0.9 ml
Water 42.0 ml
______________________________________
The preformed negatively charged vesicles were then mixed with an excess of
DEET and centrifuged at 3500 RPM for 30 minutes. Uptake of DEET into the
vesicles was approximately 0.4 ml DEET/ml vesicle.
Similar results have been obtained with a variety of flavor oils,
fragrances, and the hand cleaner d-limonene. In addition, the 40-50
strokes of the syringe, mixing the vesicles and the water immiscible
material, has been replaced by merely placing all the materials in a tube
and blending with a vortex mixer, stirrer, or homogenizer thereby
encapsulating the water immiscible material
Those skilled in the art may appreciate other methods which are within the
scope of the present invention. Such other methods are included within the
following claims.
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